ABSTRACT. Over 25 million people worldwide suffer from corneal blindness in one or both eyes. Corneal transplantation is the only option available to restore vision. However, there is a worldwide donor shortage, and fewer than 1% of patients with corneal blindness receive a transplant each year. Despite the great promise of corneal tissue engineering research to fill this gap, efforts to develop a clinically viable corneal substitute are hindered primarily due to the inability of the existing in vitro culture systems to reproduce the intricate extracellular matrix structure (ECM) of the stroma. Approximately 95% of the stroma is composed of type I collagen-based ECM in which regularly packed collagen fibrils have a uniform diameter and are arranged as orthogonal lamellae, providing the cornea with its unique mechanical and optical properties. The predominantly acellular nature of the stroma has motivated us to pursue a cell-free approach to the engineering of the stroma. We have recently demonstrated that the planar confinement of crowded collagen molecules induces self-organization of the collagen into highly ordered structures. Our objective here is to engineer an acellular stromal analog. Our central hypothesis is that a fully functional corneal stroma can be developed by harnessing the inherent physicochemical properties of collagen molecules. Our efforts to pursue the goals of this proposal will be pursued in three specific aims: In Aim 1, the effects of confining and crowding conditions on the long and short-range organization of collagen fibrils will be determined. Next, the impact of lumican and decorin proteoglycans on collagen ultrastructure (i.e. diameter and spacing) will be elucidated. In Aim 2, we will use a combination of theoretical and numerical modeling to provide an understanding of how crowding and confining conditions, as well as interactions with proteoglycans, impact collagen organization, morphology, and self-assembly. The predictive nature of the model would also enable identifying additional experimental parameters to further optimize the stromal-mimetic collagenous structures. In Aim 3, the long-term in vivo function of the acellular stromal analogs will be delineated by transplanting them into rabbits with deep corneal scars. In addition, in a set of exploratory studies we will investigate whether the native-like physical characteristics of the constructs (e.g., fibrillary organization, diameter and mechanical properties), would improve the differentiation of corneal stromal stem cells into native corneal stromal keratocytes. If successful, the work described here is expected to result in the development of first generation acellular corneal stromal analogs which can restore corneal function upon transplantation. The acellular stroma could be used directly for lamellar transplant in vivo or they could be integrated with the epithelial and endothelial layers to produce a fully functional cornea. Furthermore, the versatile collagen organizing technique that we propose to develop could be used for the production of biomimetic substrates to be integrated into the existing in vitro platforms to mechanistically investigate how various properties of ECM (e.g., organization and diameter) impacts cellular fate and function.